A better understanding of regulation of enzyme synthesis in the methionine pathway would benefit understanding of many other cellular functions since methionine is required not only for protein synthesis but also as a precursor of S-adenosylmethionine which is involved in a wide variety of metabolic functions, e.g., modification of DNA, posttranscriptional modification of tRNA, fatty acid biosynthesis, and biosynthesis of spermidine. Studies are proposed to define and characterize regulatory elements in the methionine biosynthetic pathway in Escherichia coli K12. A twofold approach will be followed which involves isolation of regulatory mutants and cell-free DNA-dependent synthesis of methionine enzymes. To facilitate both approaches gene fusions will be constructed in which lacZ will be fused to the operator regions of metA, metB and metE thereby reby placing synthesis of beta-galactosidase under methionine regulation. A search for methionine regulatory mutants on the basis of beta-galactosidase constitutivity in strains carrying these fusions will be made. These mutants will be analyzed to determine their regulatory lesions. DNA from lambda phages carrying met-lac gene fusions will be used as template for cell-free synthesis of beta-galactosidase under methionine control. Cell-free enzyme synthesis will be used to characterize the regulatory elements involved and to provide an assay system for purification of the metJ geneproduct, the methionine repressor. Purified repressor protein will make possible further studies of its interaction with the operator sites for the various structural genes.